Method for constructing Parkinson mouse model based on rotenone

Abstract

The invention provides a method for constructing a Parkinson mouse model based on rotenone. The method for constructing the Parkinson mouse model based on the rotenone specifically comprises the steps that the rotenone is dissolved in sunflower seed oil through an ultrasonic heating method to obtain a rotenone solution, the rotenone solution is injected into the stomach of a mouse through an intragastric needle, intragastric administration is conducted for one month, and the Parkinson mouse model is successfully constructed. The sunflower seed oil is used for dissolving fat-soluble rotenone, and the sunflower seed oil is used as a medium to replace sodium carboxymethyl cellulose, so that interference factors of sodium cellulose and DMSO (dimethylsulfoxide) or chloroform are eliminated, the rotenone solution can be prepared more easily, and the preparation of the rotenone solution is simplified. By means of the method, the Parkinson's mouse model can be successfully modeled, the successfully modeled mouse can generate olfactory disorder, the method is easy to operate, low in cost and long in modeling period, the established Parkinson's mouse model can better simulate the disease progress of Parkinson's disease, and scientific research can be better carried out.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically a method for constructing a Parkinson's mouse model based on rotenone. Background Technology

[0002] Currently, Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD). Animal experiments are frequently required to study Parkinson's disease, and the animal models currently used include chemically induced models, genetically modified models, and protein aggregation models. Among these numerous models, chemically induced models are particularly widely used in the laboratory, including the MPTP model (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), the 6-hydroxydopamine (6-OHDA) model, and the rotenone model.

[0003] Rotenone is a naturally occurring isoflavone compound, primarily extracted from the roots of leguminous plants such as *Derris* and *Lonchocarpus*. Due to its potent insecticidal activity, it is widely used in agricultural pesticides. Initial research exploring whether rotenone could be used to model Parkinson's disease was based on its inhibition of mitochondrial respiratory chain complex I. Mitochondrial respiratory chain complex I is a key component of the cellular aerobic respiration electron transport chain, responsible for transferring electrons from NADH to coenzyme Q. Rotenone competitively binds to the coenzyme Q binding site of complex I, blocking electron transport, leading to reduced ATP production and insufficient cellular energy supply. The initial formulation of the rotenone solution involved first preparing a 50-fold concentrate of rotenone using 100% dimethyl sulfoxide (DMSO), then diluting it in medium-chain triglycerides (Miglyol 812 N) to obtain a rotenone concentration of 2.75 or 3.0 mg / mL in 98% Miglyol 812 N and 2% DMSO. The solution was then vortexed to form a stable DMSO emulsion containing rotenone and Miglyol 812 N.

[0004] With technological advancements and improvements, the commonly used method for preparing rotenone solution is as follows: 0.72 g of sodium chloride and 3.2 g of sodium carboxymethyl cellulose are dissolved thoroughly in 79 mL of ddH2O, followed by the addition of 50 mg of rotenone pre-dissolved in 1 mL of chloroform and thorough mixing. In this technique, sodium carboxymethyl cellulose acts as a thickener, making the preparation method complex. Sodium carboxymethyl cellulose is not readily soluble in water, and preparation with room-temperature water is prone to clumping; therefore, higher-temperature water is required. Furthermore, strict requirements exist regarding the amount added, ultrasonication, vortexing, and rotor stirring, making the experiment complex, difficult to prepare, prone to failure, and with a short shelf life. Additionally, because the preparation of sodium carboxymethyl cellulose requires specific water temperatures, and rotenone is prone to decomposition if directly exposed to the high-temperature environment required for its preparation, rotenone must be dissolved in DMSO or chloroform, shaken to mix thoroughly, and then the rotenone solution must be evenly distributed within the sodium carboxymethyl cellulose solution. However, rotenone still easily precipitates out after standing, so this method cannot successfully model all rats or mice. Furthermore, DMSO and chloroform are both toxic substances and their use is unsafe. Therefore, how to successfully create a Parkinson's mouse model based on rotenone remains a major challenge. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a Parkinson's mouse model based on rotenone. This method uses sunflower seed oil as a medium to dissolve rotenone for modeling, resulting in a high success rate and no toxicity. It solves the problems of low success rate and unsafe use caused by using sodium carboxymethyl cellulose and DMSO (or chloroform) to dissolve rotenone in the prior art.

[0006] This invention is implemented as follows: A method for constructing a Parkinson's disease mouse model based on rotenone involves dissolving rotenone in sunflower seed oil using an ultrasonic heating method to obtain a rotenone solution. The rotenone solution is then administered into the stomach of mice via gavage injection. After one month of gavage administration, the Parkinson's disease mouse model was successfully constructed.

[0007] In the above scheme, preferably, the concentration of rotenone solution is 1.5~3 mg / mL.

[0008] In the above scheme, preferably, the amount of medication administered by gavage is calculated based on the weight of the mouse and meets the condition of 10~20 mg / kg.

[0009] In the above scheme, the preferred ultrasonic heating method is to conduct ultrasonication for 30 minutes at an ultrasonic frequency of 50 kHz, and control the heating temperature at 30~40℃.

[0010] In the above scheme, the preferred method is to administer the medication by gavage for one month, specifically: administer the medication by gavage for six consecutive days, rest for one day, and so on, to achieve the goal of administering the medication by gavage for one month.

[0011] In the above scheme, preferably, the rotenone solution is prepared fresh for use. If there is powder in the rotenone solution, the powder is evenly distributed in the sunflower seed oil by shaking and vortexing before administration by gavage.

[0012] This invention uses sunflower seed oil to dissolve the fat-soluble rotenone, and uses sunflower seed oil as a medium instead of sodium carboxymethyl cellulose, eliminating the interference of sodium cellulose and DMSO or chloroform. This makes it easier to prepare rotenone solutions and simplifies the preparation process. The method of this invention successfully established a Parkinson's disease mouse model, facilitating subsequent research on Parkinson's disease.

[0013] The method of this invention can successfully create a Parkinson's disease mouse model and successfully induce olfactory dysfunction in the model mice. The method of this invention is simple to operate, low in cost, and has a long modeling cycle. The established Parkinson's disease mouse model can better simulate the pathogenesis of Parkinson's disease and is more consistent with the actual pathogenesis of patients. The established Parkinson's disease mouse model can be used for screening and treatment of Parkinson's disease-related pathological mechanisms or pharmacological research. Attached Figure Description

[0014] Figure 1 These are the behavioral experiment results of the Parkinson's rat model constructed based on rotenone in the embodiments of the present invention; wherein, A is the test result of the number of times the rotundus fell; B is the test result of the rotundus fall latency; C is the test result of the open field experiment; and D is the test result of the duration of grip force under wire suspension. Detailed Implementation

[0015] In this invention, rotenone is still used when creating the Parkinson's mouse model, but the medium is changed to sunflower seed oil. As a type of vegetable oil, sunflower seed oil contains polyunsaturated fatty acids, which can dissolve fat-soluble rotenone. Furthermore, as a vegetable oil, sunflower seed oil will not cause a high-fat model in Parkinson's mice during model creation.

[0016] The amount of rotenone that sunflower seed oil can dissolve is limited; beyond a certain range, rotenone will not dissolve completely. When weighing rotenone, it's best to select a dispersed powder. Large clumps of rotenone will affect the dissolution effect because large clumps have a smaller contact area with sunflower seed oil, resulting in lower solubility.

[0017] During the dissolution process, this invention employs an ultrasonic heating method to maximize the dissolution of rotenone. Ultrasound, through physical means, uses vibration to break down larger powder molecules into smaller ones. This invention uses an ultrasonic heating-assisted dissolution method, which differs from both simple ultrasonic methods and water bath methods. The drawbacks of the water bath method are that water evaporates easily, making it difficult to control the water temperature within a suitable range, and excessively high temperatures can cause rotenone decomposition. Simple ultrasonic methods, on the other hand, do not dissolve rotenone effectively. Both ultrasonic and water bath methods result in insufficient rotenone concentration, leading to the failure of the modeling experiment. The ultrasonic heating method cleverly combines the advantages of simple ultrasonic vibration and the water bath method, while simplifying them.

[0018] After dissolving rotenone using the ultrasonic heating method, if a certain amount of powder remains, it will be present at the bottom of the EP tube. Before use, the tube should be shaken thoroughly to ensure that the powder is evenly suspended within it. These undissolved powdery rotenone particles will still be administered to rats or mice via gavage, ensuring that rotenone can exert its original effect in rats or mice.

[0019] This invention simplifies the method of preparing rotenone solution using sodium carboxymethyl cellulose combined with DMSO (or chloroform) to create a Parkinson's mouse model. The only medium required is sunflower seed oil, which greatly simplifies the dissolution process of rotenone and makes the operation simple. Moreover, it can give full play to the role of rotenone itself, and there are no other factors that interfere with the creation of the Parkinson's mouse model.

[0020] The following is a detailed example illustrating the method for constructing a Parkinson's mouse model based on rotenone, as described in this invention. The experimental procedure is as follows: Experimental materials: 5mL EP tubes, rotenone, sunflower seed oil.

[0021] Regarding the preparation of rotenone solutions, transparent EP tubes were used, and experiments were conducted at three concentration gradients: 10 mg / kg, 15 mg / kg, and 20 mg / kg. Rotenone solutions were prepared according to these three dosages. Appropriate amounts of rotenone were weighed and prepared at ratios of 1.5 mg / mL, 2.25 mg / mL, and 3 mg / mL (these three concentrations correspond one-to-one with 10 mg / kg, 15 mg / kg, and 20 mg / kg, and were chosen to ensure that the stomach volume of the mouse does not exceed 0.01 mL / g). Therefore, for a 30g mouse, to achieve concentrations of 10 mg / kg, 15 mg / kg, and 20 mg / kg, 0.2 mL of rotenone solution at concentrations of 1.5 mg / mL, 2.25 mg / mL, and 3 mg / mL, respectively, would be administered.

[0022] Rotenone solutions were prepared at concentrations of 1.5 mg / mL, 2.25 mg / mL, and 3 mg / mL in sunflower seed oil. The results showed that the 1.5 mg / mL solution had the highest solubility, producing a clear solution with no visible powder. The 2.25 mg / mL solution contained fine powder at the bottom of the EP tube, which could be removed by shaking. The 3 mg / mL solution showed powder at the bottom, which remained even after shaking, but this did not affect the final results of the gavage administration experiment in Parkinson's disease-inducing mice.

[0023] This invention uses food-grade sunflower seed oil as a medium to dissolve rotenone. Rotenone is insoluble in sunflower seed oil at room temperature, and vortexing fails to dissolve it successfully. This invention uses ultrasound combined with heating to aid in the dissolution of rotenone. During dissolution, the ultrasound temperature is controlled at 30-40°C to prevent rotenone decomposition. The ultrasound frequency is 50 kHz, and the duration is 30 minutes. If powder appears at the bottom of the EP tube, it can be evenly distributed in the sunflower seed oil using vortexing. The presence of powder does not affect oral administration. Because ultrasound breaks down rotenone into small, visible molecules that can pass through the gavage needle, it can still be successfully administered into the mouse stomach to exert its effect.

[0024] Rotenone solution was prepared fresh for each use. It was administered by gavage for six days a week, followed by a one-day rest period. After one month of gavage administration, the above three concentration gradients (10 mg / kg, 15 mg / kg, and 20 mg / kg) were able to successfully establish a Parkinson's mouse model.

[0025] For the Parkinson's mouse model constructed using an experiment with a concentration of 10 mg / kg, through Figure 1 This was verified through behavioral experiments involving options A, B, C, and D. Figure 1 The robin test shown in Figures A and B reveals a significant difference between the control group and the robinone group in terms of the number of falls and the fall latency. The robinone group experienced significantly more falls and a shorter fall latency. The open field test shown in Figure C shows that the mice in the robinone group covered a shorter total distance than the control group. The wire suspension test shown in Figure D shows that the grip strength of the robinone-induced Parkinson's mice was maintained for a shorter time than that of the control group. Therefore, these experiments demonstrate that dissolving robinone in sunflower seed oil can successfully model Parkinson's disease in mice, exhibiting symptoms such as decreased motor function and bradykinesia. Furthermore, the Parkinson's mice also developed olfactory dysfunction.

[0026] Rotenone is used to model Parkinson's disease in a way that more closely resembles the natural progression of PD in humans. The rotenone model effectively mimics the core pathological and behavioral manifestations of PD, which is its key advantage for widespread application. This method allows for easier and more effective creation of animal models of Parkinson's disease, producing corresponding motor and olfactory disturbances. This enables better replication of the pathological model of Parkinson's disease and facilitates more scientific research.

Claims

1. A method for constructing a Parkinson's mouse model based on rotenone, characterized in that, Rotenone was dissolved in sunflower seed oil using an ultrasonic heating method to obtain a rotenone solution. The rotenone solution was then administered into the stomachs of mice via gavage for one month, successfully establishing a Parkinson's mouse model.

2. The method for constructing a Parkinson's mouse model based on rotenone according to claim 1, characterized in that, The concentration of rotenone solution is 1.5~3 mg / mL.

3. The method for constructing a Parkinson's mouse model based on rotenone according to claim 1, characterized in that, The dosage for oral administration should be calculated based on the mouse's body weight, meeting the condition of 10~20 mg / kg.

4. The method for constructing a Parkinson's mouse model based on rotenone according to claim 1, characterized in that, The specific ultrasonic heating method is as follows: ultrasonication is performed for 30 minutes at an ultrasonic frequency of 50 kHz, and the heating temperature is controlled at 30~40℃.

5. The method for constructing a Parkinson's mouse model based on rotenone according to claim 1, characterized in that, The administration of medication by gavage for one month is specifically: administer the medication by gavage for six consecutive days, rest for one day, and so on, to achieve the goal of administering medication by gavage for one month.

6. The method for constructing a Parkinson's mouse model based on rotenone according to claim 1, characterized in that, Rotenone solution should be prepared fresh for use. If there is powder in the rotenone solution, the powder should be evenly distributed in the sunflower seed oil by shaking and vortexing before gavage administration.

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